Preliminary Study of Scent Rolling in Captive Wolves (Canis lupus L. 1758) in Response to Olfactory Enrichment.

Scent rolling, a behaviour observed in various large carnivores like wolves, entails the animal lowering its chin and neck towards a scent, followed by rubbing the head, neck, shoulders, and back into it. This behaviour is prevalent among wolves exposed to diverse scents, though its exact purpose remains uncertain. In this study, captive wolves at Osijek Zoo responded differently to odours during olfactory enrichment sessions. In the initial year of this study, the highest level of interest, evidenced by both the frequency of responses and scent-rolling behaviour, was noted when the wolves encountered odours such as curry and rosemary, along with deer/mouflon and rat faeces. While certain odours, such as llama faeces and deer/mouflon faeces, garnered longer durations of interest in the second year of study, others, like guinea pig faeces and oregano, elicited shorter responses. Female wolves demonstrated a higher level of engagement with scents compared with males, particularly through scent rolling behaviour, which was exclusively observed in females during the second year of this study. Interestingly, certain odours did not trigger scent rolling, suggesting selective preferences. On the other hand, sheep's wool induced the longest duration of scent rolling, and a lack of significant differences in behaviour was observed between morning and afternoon sessions. Despite the existence of multiple hypotheses put forward to explain the causation of scent rolling in wolves, it seems to be elicited by unfamiliar odours.

A Dangerous Couple: Sequential Effect of Phosphorus Flame-Retardant and Polyurethane Decrease Locomotor Activity in Planarian Girardia tigrina.

Understanding the interplay among organophosphorus flame retardants (OPFRs), microplastics, and freshwater organisms is crucial for unravelling the dynamics within freshwater environments and foreseeing the potential impacts of organic pollutants and plastic contamination. For that purpose, the present research assessed the exposure impact of 10 mg L-1 flame-retardant aluminium diethylphosphinate (ALPI), 10 µg mg-1liver microplastics polyurethane (PU), and the combination of ALPI and PU on the freshwater planarian Girardia tigrina. The exposure to both ALPI and PU revealed a sequential effect, i.e., a decrease in locomotor activity, while oxidative stress biomarkers (total glutathione, catalase, glutathione S-transferase, lipid peroxidation) and metabolic responses (cholinesterase activity, electron transport system, and lactate dehydrogenase) remained unaffected. Despite this fact, it was possible to observe that the range of physiological responses in exposed organisms varied, in particular in the cases of the electron transport system, cholinesterase activity, glutathione S-transferase, catalase, and levels of total glutathione and proteins, showing that the energetic costs for detoxification and antioxidant capacity might be causing a lesser amount of energy allocated for the planarian activity. By examining the physiological, behavioural, and ecological responses of planarians to these pollutants, insights can be gained into broader ecosystem-level effects and inform strategies for mitigating environmental risks associated with OPFRs and microplastic pollution in freshwater environments.

White Stork Pellets: Non-Invasive Solution to Monitor Anthropogenic Particle Pollution.

The present study applied a non-invasive method to analyse anthropogenic particles and prey items in white stork (Ciconia ciconia) pellets. Pellets (n = 20) were obtained from white stork nests during the 2020 breeding season from two sites in Croatia. In total, 7869 anthropogenic particles were isolated. The majority of particles were fragments, while previous studies on other birds often reported fibres. An ATR-FTIR polymer analysis detected glass and construction and building materials, as well as several compounds associated with plastic masses. Polymer investigation revealed the presence of dotriacontane and octacosane, which are by-products of polyethylene (PE) degradation and transformation. Additionally, the detection of vinylidene chloride (VDC) highlights the historical contribution of polyvinylidene chloride (PVDC) to plastic pollution. Significant variation in particle quantity and size between the sampling sites was detected, with larger particles found at sites associated with the metal mechanical engineering industry and agriculture. Prey assessment revealed chitin remains of large insects such as Orthoptera and Coleoptera. This research confirms the potential of pellet analysis as a valuable tool for assessing the presence of anthropogenic particles in the environment. However, further research is needed to fully understand the extent of particle ingestion, particle sources and potential impact.

Effects of white-tailed eagle (Haliaeetus albicilla) nestling diet on mercury exposure dynamics in Kopacki rit Nature Park, Croatia.

The present study assessed for the first time the magnitude and dietary ecological source of total mercury (THg) exposure in a southern population of white-tailed eagles (Haliaeetus albicilla), an apex predator species shown valuable for environmental biomonitoring. This population depends on the Kopacki rit Nature Park - the most important breeding site. We assessed THg exposure, using nestling body feathers collected between 2014-2019 (n = 72), and potential dietary ecological sources, proxied by prey remains and stable isotope analysis. Results show THg concentrations vary significantly over the years, though not showing any time trend. Prey remains analysis shows nests with aquatic prey remains to exhibit higher THg concentrations (median: 7.57 µg g-1 dw; min - max: 6.00-13.16 µg g-1 dw) compared to those with terrestrial remains (median: 3.94 µg g-1 dw; min - max: 0.28-12.04 µg g-1 dw) or evidencing a mixed diet (median: 7.43 µg g-1 dw; min - max: 3.38-12.04 µg g-1 dw). Nests with a predominant aquatic diet show elevated lower δ13C and higher δ15N values, indicating agreement between both dietary approaches. The model selection reveals a combination of year and δ15N best explain the variability in feather THg concentrations. Complementing these predictors with a dietary descriptor based on prey remains results in a poorer model fit and lowered explanatory power, similar to sexing the nestlings. The observed body feather THg concentrations (median: 6.99 µg g-1 dw; min - max: 0.27 - 17.16 µg g-1 dw) exceeded putative biogeochemical background levels (5.00 µg g-1 dw) in 71% of the nestlings, though, did not seem to exceed a threshold at which detrimental physiological effects are expected (40 µg g-1 dw). Continued monitoring is warranted as the studied population is likely exposed to a larger cocktail of contaminants while resident-protected bird areas.

Heavy metal(loid) effect on multi-biomarker responses in apex predator: Novel assays in the monitoring of white stork nestlings.

The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.

Blood biomarkers in white stork (Ciconia ciconia) nestlings show different responses in several areas of Croatia.

White stork nestlings can provide quantitative data on the quality of the environment, as they are dependent on their parents that provide locally foraged food. Blood was sampled from the brachial vein (n = 109) and the sampling was performed in parallel with ringing during breeding season 2020 from five areas in eastern Croatia: Lonjsko polje, Jelas polje, Slavonski Brod-east, Podunavlje, and Donje Podravlje. In the present study, for the first time in Croatia, the following enzymatic biomarkers were assessed in white stork nestlings: activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as nonenzymatic biomarkers: levels of glutathione (GSH) and reactive oxygen species (ROS). All endpoints were measured in two blood fractions: plasma and a postmitochondrial fraction (S9). Nestlings from Podunavlje and Donje Podravlje, areas known for intensive agriculture, showed lower AChE and CES activity when compared to the other investigated areas, indicating the presence of inhibitory xenobiotics. Higher oxidative stress was observed in Slavonski Brod-east, an area surrounded by metal and engineering industry, and Podunavlje compared to the other sampling areas. Hence, this study shows the impact of pollutants from the surrounding metal, petroleum, and agricultural industry might have on the biomarkers in white stork nestlings, which are often seen as early-warning signals.

Application of Non-Destructive Methods: Biomarker Assays in Blood of White Stork (Ciconia ciconia) Nestlings.

White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings' physiological condition, biomarkers can be measured in plasma and S9.